This proposal will investigate the hypothesis that ephrins and their associated Eph receptor tyrosine kinases regulate inhibitory cell-cell interactions that mediated segregation of glial cells and meningeal fibroblasts, induce glial scar formation, and inhibit axonal regeneration following spinal cord lesions. To address these questions, we will perform over-hemisection lesions of the thoracic spinal cord in adult rats and mice with gene deletions or mutations of specific ephrins or Eph receptors. Additional in vitro studies will evaluate the effects of cytokines/growth factors and proteins that block or stimulate signaling between ephrins and Eph receptors on glial cell and meningeal fibroblast functions. A variety of neuroanatomical, biochemical and molecular techniques will be used to address three specific aims. Aim I will test the hypothesis that changes in the levels and activity state of Eph/ephrins after spinal cord injury regulate cell intermixing, glial scar formation, and the reformation of the glial limitans. Cell types expressing specific Eph/ephrins will be identified and their location correlated with cellular changes in the glial scar and the distribution of inhibitory CSPGs. Aim II will use in vitro preparations to test the hypotheses that cytokineslgrowth factors regulate expression of Eph/ephrins in CNS glia and meningeal fibroblasts and that signaling through specific ephrins and Eph receptors mediates astrocyte-meningeal cell segregation and boundary formation. In these studies cytokines/trophic factors, ephrin/Eph-Fc proteins and antisense oligonucleotides will be used to regulate the production of individual ephrins/Eph receptors or alter their activation to identify their functions. Cultures of primary cells from mice with deletions and/or mutations of specific Eph/ephrins also will be used to identify the functions of specific molecules. Aim III will test the hypothesis that interactions between Eph receptors expressed on corticospinal axons and ephrins present on reactive glia in the lesioned spinal cord inhibit axonal regeneration. In these studies bilateral dorsal funiculus lesions in mice with deletions and/or mutations in ephrin-B3 or EphA4 and rats with fetal spinal cord transplants and intraspinal infusions of ephrin/Eph-Fc proteins will be used to study axonal regeneration from lesioned corticospinal axons. These experiments will permit us to investigate whether disrupting Eph-ephrin signaling enhances regeneration through the lesion/transplant interface and into the distal spinal cord. Thus, the proposed studies will expand our knowledge of the function of Eph/ephrins beyond current assumptions that they primarily regulate compartment formation and axonal guidance during nervous system development. It is anticipated that these studies also will provide important information for developing strategies to treat human spinal cord injuries.